中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (11): 1758-1764.doi: 10.12307/2023.143

• 皮肤粘膜组织构建 skin and mucosal tissue construction • 上一篇    下一篇

miR-382-3p抑制人增生性瘢痕成纤维细胞的增殖

贺  茜1，2，3，马  芳2，4，万  瑀1，2，3，唐玉婷1，2，3，马胜超2，4，姜怡邓2，4，沈江涌3   

  1. 宁夏医科大学，1临床医学院，4基础医学院，宁夏回族自治区银川市  750004；2国家卫生健康委代谢性心血管疾病研究重点实验室，宁夏回族自治区银川市  750004；3宁夏医科大学总医院烧伤整形外科，宁夏回族自治区银川市  750004
  • 收稿日期:2022-04-23 接受日期:2022-05-30 出版日期:2023-04-18 发布日期:2022-09-26
  • 通讯作者: 沈江涌，硕士，副教授，宁夏医科大学总医院烧伤整形外科，宁夏回族自治区银川市 750004
  • 作者简介:贺茜，女，1995年生，湖南省湘潭市人，汉族，宁夏医科大学在读硕士，主要从事增生性瘢痕形成的机制研究。
  • 基金资助:
    国家自然科学基金(81860555)，项目负责人：沈江涌；宁夏高等学校一流学科建设(宁夏医科大学国内一流建设学科临床医学)资助项目(NXYLXK2017A05)，项目负责人：沈江涌

miR-382-3p inhibits the proliferation of human hypertrophic scar fibroblasts

He Xi1, 2, 3, Ma Fang2, 4, Wan Yu1, 2, 3, Tang Yuting1, 2, 3, Ma Shengchao2, 4, Jiang Yideng2, 4, Shen Jiangyong3   

  1. 1School of Clinical Medicine, 4School of Basic Medicine, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 2State Key Laboratory of Metabolic Cardiovascular Disease, National Health Commission of China, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 3Department of Burn Plastic Surgery, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • Received:2022-04-23 Accepted:2022-05-30 Online:2023-04-18 Published:2022-09-26
  • Contact: Shen Jiangyong, Master, Associate professor, Department of Burn Plastic Surgery, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • About author:He Xi, Master candidate, School of Clinical Medicine, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; State Key Laboratory of Metabolic Cardiovascular Disease, National Health Commission of China, Yinchuan 750004, Ningxia Hui Autonomous Region, China; Department of Burn Plastic Surgery, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81860555 (to SJY); First-class Discipline Construction Project in Ningxia Colleges and Universities (Construction of Domestic First-Class Discipline Clinical Medicine in Ningxia Medical University), No. NXYLXK2017A05 (to SJY)

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摘要：

文题释义：
微小RNA(microRNA，miRMA)：是一种小的寡核苷酸非编码RNA，它可参与很多生物学过程，例如细胞的增殖、分化、凋亡等。
细胞增殖：是细胞进行分裂，一个细胞变成多个细胞的表现。

背景：目前多项研究证实了miRNA影响增生性瘢痕的发生发展，STAT1参与瘢痕成纤维细胞的增殖过程，猜测miR-382-3p也有可能与增生性瘢痕的发生发展有关系。
目的：探讨miR-382-3p对人增生性瘢痕成纤维细胞增殖的作用机制。
方法：收集宁夏医科大学总医院烧伤整形外科提供的增生性瘢痕和同一个体正常皮肤，提取人增生性瘢痕成纤维细胞和人正常皮肤成纤维细胞；细胞转染分别设置：①对照组(不做处理)、miR-382-3p阴性对照组、miR-382-3p过表达组；②对照组(不做处理)、STAT1干扰对照组(si-NC)和STAT1干扰组(si-STAT1-1、si-STAT1-2、si-STAT1-3)。苏木精-伊红染色鉴定正常皮肤和增生性瘢痕；免疫荧光鉴定成纤维细胞；qRT-PCR检测miR-382-3p、STAT1、增殖细胞核抗原及细胞周期依赖性激酶抑制物(p27)mRNA表达；Western blot检测STAT1、增殖细胞核抗原及p27蛋白表达；CCK8、EdU检测细胞增殖活力和增殖水平；Targetscan预测miR-382-3p的下游靶基因，双荧光素酶验证miR-382-3p与STAT1的结合情况。
结果与结论：①与正常皮肤和正常皮肤成纤维细胞相比，miR-382-3p在增生性瘢痕和增生性瘢痕成纤维细胞中呈低表达(组织：P < 0.01，细胞：P < 0.01)，STAT1在增生性瘢痕和增生性瘢痕成纤维细胞中呈高表达(组织水平mRNA：P < 0.01，组织水平蛋白：P < 0.01；细胞水平mRNA：P < 0.01，细胞水平蛋白：P < 0.01)；②过表达miR-382-3p后细胞增殖能力减弱(P < 0.05)，EdU阳性细胞数减少(P < 0.01)，增殖细胞核抗原的表达减少(mRNA：P < 0.01，蛋白：P < 0.05)，p27的表达增加(mRNA：P < 0.05，蛋白：P < 0.05)；③miR-382-3p能够靶向调控STAT1的表达(P < 0.01)，过表达miR-382-3p可导致STAT1的表达减少(mRNA：P < 0.01，蛋白：P < 0.01)；④干扰STAT1后可导致增殖细胞核抗原的表达减少(P < 0.05)，p27的表达增加(P < 0.05)，EdU阳性细胞数减少(P < 0.01)；⑤结果表明：miR-382-3p可通过抑制STAT1的表达进而抑制增生性瘢痕成纤维细胞的增殖，为未来增生性瘢痕的治疗寻找有效靶点提供一定的理论依据。

https://orcid.org/0000-0001-7859-0221(贺茜)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: miR-382-3p, STAT1, PCNA, p27, 增生性瘢痕, 正常皮肤, 成纤维细胞, 增殖

Abstract: BACKGROUND: At present, numerous studies have shown that miRNA is involved in the occurrence and development of hypertrophic scars and STAT1 is involved in the proliferation of scar fibroblasts. It is speculated that miR-382-3p may also be related to the occurrence and development of hypertrophic scars.
OBJECTIVE: To investigate the mechanism of miR-382-3p on the proliferation of human hypertrophic scar fibroblasts.
METHODS: Hypertrophic scar and normal skin of the same individual were collected from the Department of Burn Plastic Surgery, General Hospital of Ningxia Medical University, and human hypertrophic scar fibroblasts and human normal skin fibroblasts were then extracted. Cell transfection was conducted as follows: (1) the cells were divided into control group (no treatment), miR-382-3p negative control group, and miR-382-3p overexpression group; (2) the cells were divided into control group (no treatment), STAT1 interference control group (si-NC) and STAT1 interference groups (si-STAT1-1, si-STAT1-2, si-STAT1-3). Hematoxylin-eosin staining was used to identify normal skin and hypertrophic scar, and immunofluorescence was used to identify fibroblasts. Quantitative real-time PCR was applied to detect the mRNA expression of miR-382-3p, STAT1, proliferating cell nuclear antigen (PCNA) and cyclin-dependent kinase inhibitor (p27). Western blot assay was performed to detect the protein expressions of STAT1, PCNA and p27. Cell counting kit-8 and EdU were used to detect cell proliferation activity and proliferation level. Targetscan was used to predict the downstream target genes of miR-382-3p and dual luciferase was used to verify the binding of miR-382-3p to STAT1.
RESULTS AND CONCLUSION: Compared with normal skin and normal skin fibroblasts, miR-382-3p was lowly expressed in hypertrophic scar and hypertrophic scar fibroblasts (tissue: P < 0.01, cell: P < 0.01), and STAT1 was highly expressed in hypertrophic scar and hypertrophic scar fibroblasts (mRNA level in tissue: P < 0.01, protein level in tissue: P < 0.01; mRNA level in cells: P < 0.01, protein level in tissue: P < 0.01). After overexpression of miR-382-3p, the cell proliferation ability was weakened (P < 0.05), the number of EdU positive cells was decreased (P < 0.01), the expression of PCNA was decreased (mRNA: P < 0.01, protein: P < 0.05), and the expression of p27 was increased (mRNA: P < 0.05, protein: P < 0.05). miR-382-3p could target and regulate the expression of STAT1 (P < 0.01), and overexpression of miR-382-3p could reduce the expression of STAT1 (mRNA: P < 0.01, protein: P < 0.01). Interference with STAT1 reduced the expression of PCNA (P < 0.05), increased the expression of p27 (P < 0.05), and reduced the number of EdU positive cells (P < 0.01). To conclude, miR-382-3p could inhibit the proliferation of hypertrophic scar fibroblasts by inhibiting the expression of STAT1, which provides a certain theoretical basis for identifying effective targets for the treatment of hypertrophic scars.

Key words: miR-382-3p, STAT1, PCNA, p27, hypertrophic scar, normal skin, fibroblast, proliferation

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